Field of the Invention
This invention relates to a method that uses surface plasmon field enhanced fluorescence spectroscopy (SPFS) and isotachophoresis (ITP) to achieve ultra-rapid and highly-sensitive biological molecules detection.
Description of Related Art
Surface plasmon field-enhanced fluorescence spectroscopy (SPFS) is a known biosensing technology. See T. Liebermann, W. Knoll, Surface-plasmon field-enhanced fluorescence spectroscopy, Colloids and Surfaces A: Physicochem. Eng. Aspects 171 (2000) 115-130 (“Liebermann 2000”); Wolfgang Knoir, Fang Yu, Thomas Neumann, Lifang Niu, and Evelyne L. Schmid, Principles And Applications Of Surface Plasmon Field-Enhanced Fluorescence Techniques, in Topics in Fluorescence Spectroscopy, Volume 8: Radiative Decay Engineering, Edited by Geddes and Lakowicz, Springer Science+Business Media, Inc., New York, 2005, p. 305-332. These references are incorporated by reference in their entireties to show the principle and setup of SPFS biosensors in general. SPFS offers high-sensitivity detection through advanced sensing technology.
FIG. 1A of this application, taken from FIG. 5 of the Liebermann 2000 paper, illustrates the setup of an SPFS system. FIG. 1B of this application, taken from FIG. 6(a) of the same paper, illustrates the structure of the prism and flow cell used in the SPFS system. The basic concept of SPFS is described below with reference to FIGS. 1, 1A and 1B. An SPFS biosensor includes a thin metal film on a glass or plastic prism. The metal may be, for example, gold, silver, aluminum, etc. A capture molecule is immobilized on the surface of the metal film. A biological sample is applied on the metal film. When an incident light of a certain wavelength is irradiated on the prism at a certain angle, a strong electrical field is generated at the surface of the metal film. Because of quenching from the metal film, the best place for fluorescence excitation is in the region about a couple of tens to hundreds nm above the surface. In a typical device, the quenching region is within about 0-5 nm from the metal surface, and the enhanced region is about 10-200 nm from the surface. If a fluorescent label is trapped in this enhanced region, strong fluorescent signal is generated.
SPFS biosensors are based on fluorescence detection. In conventional SPFS biosensors, in addition to first antibodies that are immobilized on the thin metal film, fluorescent labeled second antibodies are generally used for protein detection. This is schematically illustrated in FIG. 1. The first antibodies 101 are immobilized on the thin metal film. The target 102 (i.e. substance to be detected, such as a protein) is added to the biosensor and captured on the immobilized first antibodies. Then, the fluorescent labeled second antibodies 103 are added to the biosensor and they bind to the target. The first antibody 101, the target 102 and the second antibody 103 form a structure such that the fluorescent label 103F on the second antibody is located in the region of enhanced electric field above the thin metal film, and a strong fluorescent signal is generated. For unbound second antibodies or those that form non-specific binding, their fluorescent labels tend to be located outside of the enhanced region, either in the metal quenching region or farther away from the surface, so they are not excited. The biosensor can be washed before the detection result is obtained. These multiple steps make the biosensor more complicated to use and the turnaround time long.
PCT application WO 2011155435 A1, Near field-enhanced fluorescence sensor chip, also describes surface plasmon field enhanced fluorescence spectroscopy.
Isotachophoresis (ITP) is an electrophoresis technique that uses two buffers including a high-mobility leading electrolyte (LE) and a low-mobility trailing electrolyte (TE). In peak-mode ITP, sample species bracketed by the LE and TE focus into a narrow TE-to-LE interface by application of an electric field of typically a few hundred volts per cm. Due to the high concentration of sample species in a small volume at the interface, high efficiency (rapid) molecular-molecular interaction can occur. ITP has been used, for example, to selectively extract and concentrate medically relevant biomarkers from body fluids such as whole blood and urine sample.
An ultra-rapid nucleic acid detection technology using ITP is described in Rapid Detection of Urinary Tract Infections Using Isotachophoresis and Molecular Beacons, M. Bercovici et al., Analytical Chemistry 2011, 83, 4110-4117 (“Bercovici et al. Analytical Chemistry 2011”). This method accelerates DNA hybridization by using ITP. FIG. 1 of this article, reproduced as FIG. 2 of the instant disclosure, shows the principle of detection. The article describes: “FIG. 1a schematically presents the principles of the assay. ITP uses a discontinuous buffer system consisting of LE and TE, which are typically chosen to have respectively higher and lower electrophoretic mobility than the analytes of interest. Both sample and molecular beacons are initially mixed with the TE. When an electric field is applied, all species with mobility higher than that of the TE electromigrate into the channel. Other species (including ones with lower mobility, neutral or positively charged) remain in or near the sample reservoir. Focusing occurs within an electric field gradient at interface between the LE and TE, as sample ions cannot overspeed the LE zone but overspeed TE ions.” (Id., p. 4111, left column.) “FIG. 1. (a) Schematic showing simultaneous isotachophoretic extraction, focusing, hybridization (with molecular beacons), and detection of 16S rRNA bound to a molecular beacon. Hybridization of the molecular beacon to 16S rRNA causes a spatial separation of its fluorophore and quencher pair resulting in a strong and sequence-specific increase in fluorescent signal. (b) Raw experimental image showing fluorescence intensity of molecular beacons hybridized to synthetic oligonucleotides using ITP. (c) Detection of oligonucleotides having the same sequence as the target segment of 16S rRNA. Each curve presents the fluorescence intensity in time, as recorded by a point detector at a fixed location in the channel (curves are shifted in time for convenient visualization). 100 pM of molecular beacons and varying concentrations of targets were mixed in the trailing electrolyte reservoir. The total migration (and hybridization) time from the on-chip reservoir to the detector was less than a minute.” (Id., p. 4111, right column.) A setup for the on-chip ITP assay using a microfluidic chip is shown in FIGS. 2A and 2B of the instant disclosure, reproduced from FIGS. 2 and 3(a) of the above article. Han, C. M., Katilius, E., Santiago, J. G., “Increasing hybridization rate and sensitivity of DNA microarrays using isotachophoresis,” Lab on a Chip 2014 discloses a method to increase hybridization between immobilized DNA probe and free DNA by ITP.
DNAzymes are DNA molecules that have the ability to catalyze specific chemical reactions. As nucleic acids, DNAzymes offer several advantages to enzymes, including increased thermal stability and pH resistance. They have been shown to be very specific, capable of differentiating between targets differing by as little as a single nucleotide. One application of DNAzymes is in the fluorescent detection of nucleic acid targets. Y. V. Gerasimova, E. Cornett, and D. M. Kolpashchikov, “RNA-Cleaving Deoxyribozyme Sensor for Nucleic Acid Analysis: The Limit of Detection”, Chembiochem (2010), 11, 811-817 describes an assay in which a two-stranded DNAzyme is used to catalyze a reaction between a substrate internally labeled with both a fluorophore and quencher, and a 20-nucleotide DNA target. Both the substrate and the target are complementary to different sections of the DNAzyme. When the two strands of the DNAzymes, the substrate, and the target come together into one large complex, the DNAzyme cleaves the substrate, separating the fluorophore from the quencher and resulting in a signal increase. Once cleaved, the DNAzyme and target are free to react with another substrate, leading to signal amplification. This article demonstrated a 0.1 nM limit of detection, but the assay time was over 3 h.